三种用于透明质酸检测的融合蛋白的构建表达及检测效果比较  

Construction and comparison of three fusion proteins for hyaluronic acid assay

作　　者：段宁骏[1,2] 吕万胜[1,2] 贡佳 郑伟娟[1,2] 华子春[1,2] 

机构地区：[1]南京大学生命科学学院,南京210023 [2]医药生物技术国家重点实验室,南京210023

出　　处：《南京大学学报（自然科学版）》2015年第5期1084-1090,共7页Journal of Nanjing University（Natural Science）

基　　金：国家自然科学基金(81072712;81373400);江苏省自然科学基金(BK2012710)

摘　　要：透明质酸是一种长链大分子糖胺聚糖,存在于各组织器官中,是细胞外基质的重要组成部分.透明质酸不仅能够与其他胞外多糖及蛋白构成骨架,参与组织器官的结构形成,也可以与细胞表面相关蛋白受体相互作用,通过相关信号通路影响细胞的生存、增殖以及迁移.研究证实,透明质酸与肿瘤细胞的生存、迁移,肿瘤血管发生等均有密切关系,组织中透明质酸含量的变化也与某些病理状况如慢性炎症相关.因此,细胞表面或组织中的透明质酸含量的定量检测,对于疾病的诊断及预后都具有重要意义.目前透明质酸的检测方法主要依靠抗体,通过酶联免疫检测体液中的透明质酸,而缺乏在细胞或组织水平上检测透明质酸的手段.本研究将三种人源的透明质酸结合蛋白(P32,CDC37以及RHAMM)与绿色荧光蛋白(EGFP)进行融合,在大肠杆菌中进行可溶性表达,经亲和纯化,获得了三种能够与透明质酸结合的蛋白探针.通过在溶液与细胞水平上比较与透明质酸结合的能力,从中筛选出了最适合用来进行透明质酸检测的荧光蛋白探针RHC-EGFP.Hyaluronic acid（HA）is a long chain glycosaminoglycan with large molecular weight,existing in all kinds of tissue and organs as an important component of extracellular matrix（ECM）.HA can not only form scaffold for tissue and organs when combined with other glycosaminoglycan and proteins but also influence cell survival,proliferation and migration through distinct signal ways.Some research has provided that HA has strong relationship with the survival of carcinoma cells,angiogenesis in tumor and other pathological phenomenon.Therefore,quantification of HA level on cell surface or in tissue samples has great meaning in judging the developing level and prognosis of canc-er.Up to now,the main method in HA detection is Enzyme-Linked Immunosorbent Assay（ELISA）which can only get HA level in serum but not on cell surface or in tissue samples.We constructed three fusion proteins which combined green fluorescence protein with three hyaluronic acid binding protein from human（P32,CDC37 and RHAMM）,and expressed them in E.coli in a soluble form.Purified by affinity chromatography,three hyaluronic acid binding protein probe were gotten from cell lysate.Compared their hyaluronic acid binding abilities in solution and cell level,we selected the fittest protein probe RHC-EGFP for hyaluronic acid assay.

关 键 词：透明质酸 透明质酸结合蛋白 绿色荧光蛋白 检测 

分 类 号：TP181[自动化与计算机技术—控制理论与控制工程]